Natural RNA tags added to RNA of interest allow detection, characterization, labeling and affinity purification of RNA target molecules. For example, small aptamer RNAs that bind with high affinity and specificity to Sephadex beads or streptavidin allow purification of intact RNA-protein complexes. Furthermore, RNA tags inserted into selected locations in genes encoding RNA components enable their detection. The CRISPR Cas system can be used together with synthetic RNA mimics to incorporate RNA tags into specific genomic locations to allow labeling.
Recently a two-color CRISPR labeling system was developed by Wang and coworkers. Adding selective tags to a target-specific single guide RNA allows recruiting CRISPR-Cas systems for controlling gene expression.
Synthetic tagged RNA constructs allow purification of target RNAs and RNA complexes including miRNA and lncRNA as well as other molecules attached to the RNAs using pull-down techniques. RNA tags are unique tools for studying a variety of target RNAs including RNA transcripts, miRNAs, ncRNAs, lncRNAs, splicing event as well as others.
Synthetic RNA can be chemically tagged or modified through the incorporation of modified ribonucleotides, such as BNAs, functional moieties such as biotin, fluorescent dyes, or natural or synthetic aptamers, for example, S1, D8, MS2 hairpin loops. Other types of RNA or constructs can be added as well.
RNA affinity tags allow rapid enrichment of RNA binding protein (RNP) complexes from cellular lysates using mild conditions. This approach allows purification of RNP complexes under native conditions.
Also, RNA tags can be used for the isolation of precursor or other RNA molecules of interest including RNPs.
Known methods for the tagging of RNAs are:
(1) Chemical tagging during in vitro transcription.
(2) Incorporation of a well-characterized protein-binding RNA sequence
during in vitro or in vivo transcription.
(3) Hybridization of affinity-tagged oligonucleotides that can also be biotinylated or
modified, for example with BNAs.
(4) Incorporation of an artificially selected RNA motif during in vivo or
in vitro transcription.
However, each of these methods has advantages and disadvantages.
A variety of other RNA based applications are possible as well. Combining two or more hairpins within one RNA molecule is also possible.
Table 1: Common RNA Tags
Tag | Sequence Info | Notes |
Sephadex Tag | D8 Sephadex RNA motif is recognized by Sephadex.
5′ UCCGAGUAAU UUACGUUUUG AUACGGUUGC GGA 3′ | The D8Sephadex-binding RNA minimal motif has 33 nucleotides. The indicated minimal structural motif has been discovered. The D8 tag was shown to bind specifically to Sephadex G-100 (Pharmacia). The D8 tag does not bind to other similar matrices such as Sepharose or Seph-acryl. binding of the tag to Sephadex can be efficiently competed with dextran B512. |
Steptavidin Tag | S1 Streptavidin RNA motif recognized by streptavidin.
5′ ACCGACCAGA AUCAUGCAAG UGCGUAAGAU AGUCGCGGGC CGGG 3′
Kd of ~70 nM. | The S1 streptavidin-binding RNA motif has 44 nucleotides originally selected to bind to streptavidin in either streptavidin–agarose bead assays or polyacrylamide gel electrophoretic mobility shift assays. Bound RNA tags can thus be released from streptavidin under otherwise native binding conditions by the inclusion of biotin in the binding buffer. |
MS2 Tag or MS2-TRAP | Tagged RNA affinity purification. RNA is tagged with MS2 RNA hairpins and a fusion protein recognizing the MS2 RNA hairpins, MS2-GST is used. | Identification of miRNAs associated with a target transcript in the cellular context. Identification of microRNAs that associate with a long intergenic (li)ncRNA. |
BoxB sequence = Boxb Rna | NGTTCACCTCTAACCGGGTGAGCC Is recognized by the bacteriophage protein λ N: N Peptide: ESKGTAKSRYKARRAELIAERR | BoxB can be used to tether proteins to RNAs, for example to mRNAs. |
PP7 Hairpin | Binds to PP7 coat protein. | Different RNA hairpins are rcognized by the coat proteins of different single-stranded RNA phages. |
Proposed and solved Structures of Hairpins
Figure 1: Minimal binding motif and consensus structures of the Sephadex-affinity tag.
Figure 2: Minimal binding motif and consensus structures of the streptavidin-affinity tag. (X indicates nonconserved nucleotides).
Figure 3: Different views of the structural model of a MS2-RNA hairpin (G-5) complex. (Source: PDB 2C51).
Figure 4: Different views of the structural model of the MS2-RNA hairpin (G-5). (Source: PDB 2C51).
pdb|1NYB|A Chain A, Solution Structure Of The Bacteriophage Phi21
N Peptide-Boxb Rna Complex ESKGTAKSRYKARRAELIAERR
pdb|1NYB|B Chain B, Solution Structure Of The Bacteriophage Phi21
N Peptide-Boxb Rna Complex NGTTCACCTCTAACCGGGTGAGCC
Figure 5: Two views of the structural models from the solution structure of a 22-amino-acid peptide from the amino-terminal domain of the bacteriophage φ21 N protein in complex with its cognate 24-mer boxB RNA hairpin solved with NMR.
The nut (N utilization) site of bacteriophage lambda consists of two genetically defined elements, boxA and boxB. boxB forms an RNA hairpin and its 5 bp stem and 5 nt loop are recognized by the N peptide. The N peptide binds as an α-helix and interacts predominately with the major groove side of the 5′ half of the boxB RNA stem-loop. The φ21 boxB loop (CUAACC) has a structure typical of the “U-turn” motif.
Figure 6: Two views of the PP7 coat protein dimer in complex with RNA hairpin. The RNA hairpin binds across the β-sheet surface of the coat protein dimer.
The combined use of the MS2 hairpin with the PP7 hairpin allows detection of RNA in live cells if a chimeric protein consitent of the phage protein, a nuclear localization signal, and a fluorescent molecules is used. The MS2/PP7 appraoch has been used for the study of movement and localization of RNA as well the formation of RNA at the transcription site.
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